Hif1-a and mRNA Expression in Cell Cycle Regulators

Description

This project investigated the role of the hypoxia-inducible factor, HIF-I alpha, gene in mRNA expression after exposure to radiation. A profile of cell cycle regulators was made using mouse sarcoma cells.

Role of HIF-I alpha in mRNA Expression: Profile of Cell Cycle Regulators in Mouse Sarcoma Cells after Ionizing Radiation Grace Guo North Carolina School of Science and Mathematics Durham, NC

Introduction and Goals • Examine radiation’s effects on cancerous soft tissue sarcoma • Investigate how hypoxia-inducible factor-1α (HIF-1α) regulates mRNA expression of cell cycle regulators that include tumor promoters and suppressors • Use tumor samples with and without HIF-1α gene to research how the gene plays a role in mRNA expression in various cell cycle regulators

Background • • • • Genes are essential mechanisms for controlling cancer. Hypoxia-inducible factor-1α (HIF-1α): involved in the induction of oxygen regulated genes. Is a heterodimeric transcription factor consisting of an alpha (α) and a beta (β) subunit (Goda, 2003). Cyclin-dependent kinase inhibitor (CDKN) genes are a family of regulators that control cell cycle progression through proteins that function at specific phases of the cell cycle (Nowsheen, 2012) Complex damage response pathway: regulates known responses such as cell-cycle arrest and apoptosis (Zhou, 2000)

CDKs and Cyclins by Cell Cycle Phase Science Watch • Cyclin-dependent kinases (CDKs) and cyclins (ccn) were used to identify time points after irradiation where the genes are induced the most or least • Proteins use different mechanisms to control cell cycle, but are similar and not specific for a particular phase of the cycle • For comparison’s sake, CDK’s cyclins, such as ccna1-1 and ccnb1-1, were also used as specific primers to analyze gene expression.

Methods • Designed appropriate primers for genes to be tested for • One set of tumor samples had the gene HIF-1a (P7NP). Another set did not (P7NPH1). • Both irradiated. RNA samples were collected at 00 hours, 02 hours, 24 hours, and 48 hours • Used RNA/Reverse Transcriptase and cDNA protocols  complement DNA • Performed rtPCRs (real-time Polymerase Chain Reaction)

Results • Analyzed results • • • • • • Down-regulation? Up-regulation? Fold change (measure of level of gene expression) in rtPCR relative to non-irradiated control over time Used α= 0.05 significance level All Arf and Ink4a genes were up-regulators and had notable fold change. Many cell cycle regulatory factors did not have changes in the mRNA level. Would have be surprising if there were drastic changes everywhere or if there no changes at all, since P7NPH1 tumors are sensitized to irradiation

Conclusion • Most genes did not have significant mRNA expression changes before and after irradiation, with the exception of Ink4a and Arf. • Ink4a and Arf had significant mRNA expression changes • Disadvantage of RNA studies: protein translation does not occur exactly the same way or at the same speed across all proteins. • Even if there was no RNA difference it does not mean that there was no protein expression level difference.

Future Research • • • • • Examine specific cell cycle stages and the expression of mRNA during those stages Why Ink4a and Arf increase expression and what role they play Investigate cell cycle regulators at the RNA and protein levels (western blotting), not just at the mRNA levels Quantify percentage of expression in each cell cycle phase Experiment with cell senescence, or permanent cell-cycle arrest, to understand how cells react to stress and damage from outside sources

Acknowledgements I would like to thank the following people for their help: • The Kirsch lab, Duke University • Mentors: Dr. David Kirsch, Minsi Zhang, Lixia Luo. Duke University • Dr. Shoemaker, research coordinator. NC School of Science and Math.

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